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An automated growth enclosure for metabolic labeling of Arabidopsis thaliana with 13C-carbon dioxide - an in vivo labeling system for proteomics and metabolomics research.

Chen WP, Yang XY, Harms GL, Gray WM, Hegeman AD, Cohen JD - Proteome Sci (2011)

Bottom Line: Arabidopsis was grown in the enclosure for up to 8 weeks and obtained on average >95 atom% enrichment for small metabolites, such as amino acids and >91 atom% for large metabolites, including proteins and peptides.The capability of this labeling system for isotope dilution experiments was demonstrated by evaluation of amino acid turnover using GC-MS as well as protein turnover using LC-MS/MS.Because this 'open source' Arabidopsis 13C-labeling growth environment was built using readily available materials and software, it can be adapted easily to accommodate many different experimental designs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticultural Science, University of Minnesota, Saint Paul, USA. hegem007@umn.edu.

ABSTRACT

Background: Labeling whole Arabidopsis (Arabidopsis thaliana) plants to high enrichment with 13C for proteomics and metabolomics applications would facilitate experimental approaches not possible by conventional methods. Such a system would use the plant's native capacity for carbon fixation to ubiquitously incorporate 13C from 13CO2 gas. Because of the high cost of 13CO2 it is critical that the design conserve the labeled gas.

Results: A fully enclosed automated plant growth enclosure has been designed and assembled where the system simultaneously monitors humidity, temperature, pressure and 13CO2 concentration with continuous adjustment of humidity, pressure and 13CO2 levels controlled by a computer running LabView software. The enclosure is mounted on a movable cart for mobility among growth environments. Arabidopsis was grown in the enclosure for up to 8 weeks and obtained on average >95 atom% enrichment for small metabolites, such as amino acids and >91 atom% for large metabolites, including proteins and peptides.

Conclusion: The capability of this labeling system for isotope dilution experiments was demonstrated by evaluation of amino acid turnover using GC-MS as well as protein turnover using LC-MS/MS. Because this 'open source' Arabidopsis 13C-labeling growth environment was built using readily available materials and software, it can be adapted easily to accommodate many different experimental designs.

No MeSH data available.


Sealed enclosure for whole-plant labeling with 13CO2. (A) Diagram of the system, including in-line gas flow control, continuous CO2 measurement, Peltier-based dehumidifier, ethylene scrubber, sensors for temperature, humidity and enclosure pressure and data acquisition and control devices. (B) Photograph of the system placed on its movable cart. The enclosure (30.4 cm × 30.4 cm × 30.4 cm) shown is in its primary single cube configuration. An additional cube can be added on the top of the first cube to increase the total growth overhead space. (C) Three-week-old Arabidopsis seedlings grown in the enclosure from seed in 99 atom% 13CO2.
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Figure 1: Sealed enclosure for whole-plant labeling with 13CO2. (A) Diagram of the system, including in-line gas flow control, continuous CO2 measurement, Peltier-based dehumidifier, ethylene scrubber, sensors for temperature, humidity and enclosure pressure and data acquisition and control devices. (B) Photograph of the system placed on its movable cart. The enclosure (30.4 cm × 30.4 cm × 30.4 cm) shown is in its primary single cube configuration. An additional cube can be added on the top of the first cube to increase the total growth overhead space. (C) Three-week-old Arabidopsis seedlings grown in the enclosure from seed in 99 atom% 13CO2.

Mentions: Our interest in using stable isotope labeling coupled with LC-MS/MS for determination of protein and metabolite turnover necessitated the construction of an automated and versatile 13CO2 labeling enclosure. The system we designed consists of a closed growth box assembled using predominately commercially available components. The enclosure itself and the housing for the Peltier-based dehumidifier were constructed in the university shop from Plexiglass® acrylic sheets. Figure 1A and B show a schematic and photographic image, respectively, of the completed system. 13CO2 labeling using the system has been successfully tested by growing Arabidopsis as shown in Figure 1C. The controlled growth environment can accommodate 25 Arabidopsis plants seedlings grown hydroponically with a maximum head-space volume of ~50 L.


An automated growth enclosure for metabolic labeling of Arabidopsis thaliana with 13C-carbon dioxide - an in vivo labeling system for proteomics and metabolomics research.

Chen WP, Yang XY, Harms GL, Gray WM, Hegeman AD, Cohen JD - Proteome Sci (2011)

Sealed enclosure for whole-plant labeling with 13CO2. (A) Diagram of the system, including in-line gas flow control, continuous CO2 measurement, Peltier-based dehumidifier, ethylene scrubber, sensors for temperature, humidity and enclosure pressure and data acquisition and control devices. (B) Photograph of the system placed on its movable cart. The enclosure (30.4 cm × 30.4 cm × 30.4 cm) shown is in its primary single cube configuration. An additional cube can be added on the top of the first cube to increase the total growth overhead space. (C) Three-week-old Arabidopsis seedlings grown in the enclosure from seed in 99 atom% 13CO2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3046907&req=5

Figure 1: Sealed enclosure for whole-plant labeling with 13CO2. (A) Diagram of the system, including in-line gas flow control, continuous CO2 measurement, Peltier-based dehumidifier, ethylene scrubber, sensors for temperature, humidity and enclosure pressure and data acquisition and control devices. (B) Photograph of the system placed on its movable cart. The enclosure (30.4 cm × 30.4 cm × 30.4 cm) shown is in its primary single cube configuration. An additional cube can be added on the top of the first cube to increase the total growth overhead space. (C) Three-week-old Arabidopsis seedlings grown in the enclosure from seed in 99 atom% 13CO2.
Mentions: Our interest in using stable isotope labeling coupled with LC-MS/MS for determination of protein and metabolite turnover necessitated the construction of an automated and versatile 13CO2 labeling enclosure. The system we designed consists of a closed growth box assembled using predominately commercially available components. The enclosure itself and the housing for the Peltier-based dehumidifier were constructed in the university shop from Plexiglass® acrylic sheets. Figure 1A and B show a schematic and photographic image, respectively, of the completed system. 13CO2 labeling using the system has been successfully tested by growing Arabidopsis as shown in Figure 1C. The controlled growth environment can accommodate 25 Arabidopsis plants seedlings grown hydroponically with a maximum head-space volume of ~50 L.

Bottom Line: Arabidopsis was grown in the enclosure for up to 8 weeks and obtained on average >95 atom% enrichment for small metabolites, such as amino acids and >91 atom% for large metabolites, including proteins and peptides.The capability of this labeling system for isotope dilution experiments was demonstrated by evaluation of amino acid turnover using GC-MS as well as protein turnover using LC-MS/MS.Because this 'open source' Arabidopsis 13C-labeling growth environment was built using readily available materials and software, it can be adapted easily to accommodate many different experimental designs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Horticultural Science, University of Minnesota, Saint Paul, USA. hegem007@umn.edu.

ABSTRACT

Background: Labeling whole Arabidopsis (Arabidopsis thaliana) plants to high enrichment with 13C for proteomics and metabolomics applications would facilitate experimental approaches not possible by conventional methods. Such a system would use the plant's native capacity for carbon fixation to ubiquitously incorporate 13C from 13CO2 gas. Because of the high cost of 13CO2 it is critical that the design conserve the labeled gas.

Results: A fully enclosed automated plant growth enclosure has been designed and assembled where the system simultaneously monitors humidity, temperature, pressure and 13CO2 concentration with continuous adjustment of humidity, pressure and 13CO2 levels controlled by a computer running LabView software. The enclosure is mounted on a movable cart for mobility among growth environments. Arabidopsis was grown in the enclosure for up to 8 weeks and obtained on average >95 atom% enrichment for small metabolites, such as amino acids and >91 atom% for large metabolites, including proteins and peptides.

Conclusion: The capability of this labeling system for isotope dilution experiments was demonstrated by evaluation of amino acid turnover using GC-MS as well as protein turnover using LC-MS/MS. Because this 'open source' Arabidopsis 13C-labeling growth environment was built using readily available materials and software, it can be adapted easily to accommodate many different experimental designs.

No MeSH data available.